Pre-B cells in human bone marrow are destined to die unless they are rescued through survival signals from a successfully assembled pre-B cell receptor (pre-BCR). Congenital defects in pre-BCR-related signaling molecules cause a severe block of early B cell development in humans. Likewise, B cell lineage acute lymphoblastic leukemia (ALL) cells are arrested at early stages of B cell development. B cell lineage ALL represents by far the most frequent malignancy in children and is also common in adults. Despite significant advances over the past four decades, cytotoxic treatment strategies have recently reached a plateau with cure rates at ~80 percent for children and 55 percent for adults. Relapse after cytotoxic drug treatment, initial drug-resistance and dose-limiting toxicity are among the most frequent complications of current therapy approaches. For this reason, pathway-specific treatment strategies seem promising to further improve therapy options for ALL patients. The following key observations during the initial project period lay the foundation for this renewal application: ALL can be subdivided into two groups that fundamentally differ with respect to pre-BCR function. In TCF3- rearranged ALL, pre-BCR signaling enables (Type 1), but suppresses (Type 2) leukemic transformation in other cytogenetic (e.g. MLL-rearranged, Ph+) ALL subtypes (Trageser et al., J Exp Med 2009). The divergent outcome in Type 1 and Type 2 ALL is mirrored by contrasting functions of pre-BCR signaling at specific stages of normal B cell development: The pre-BCR drives proliferation of Hardy Fraction C' but differentiation and cell cycle arrest in Fraction D pre-B cells (Nahar et al., Blood 2011). In Fraction C' pre-B cells and Type 1 ALL cells, pre-BCR signaling promotes survival signaling by activation of SOX4 (Ramezani et al., Blood 2013), whereas BACH2 mediates negative selection at the pre-BCR checkpoint and tumor suppression in ALL cells (Swaminathan et al., Nature Medicine 2013). In Fraction D pre-B cells that passed the pre-BCR checkpoint, BCL6 mediates survival (Duy et al., J Exp Med 2010). Likewise, BCL6 promotes survival and a previously unrecognized form of drug-resistance in Ph+ ALL (Duy et al., Nature 2011) and self-renewal of leukemia-initiating cells in Ph+ ALL (Hurtz et al., J Exp Med 2011). Based on these and other findings, we propose three Aims for the second project period, namely to understand the mechanistic switch of pre-BCR function from proliferation to differentiation/cell cycle arrest (Aim 1), to validate pre- BCR checkpoint regulators as potential therapeutic targets in genetic loss-of-function models (Aim 2) and to leverage this information to develop a strategy for pharmacological targeting of the pre-BCR pathway and to develop biomarkers that predict outcomes and facilitate risk stratification for patients with ALL (Aim 3). The central goal of this proposal is to establish the role of pre-BCR signaling during malignant transformation and clonal evolution of ALL and to target individual components of its signaling cascade for the development of novel pathway-specific therapy approaches for ALL.